1. Field of the Invention
The present invention relates to a novel flap endonuclease mutant derived from a wild type flap endonuclease by modifying its substrate specificity, and a reagent for the analysis of genetic polymorphism using the same.
2. Background Art
A flap endonuclease is an essential enzyme for DNA replication/repair, because it recognizes DNA structure in a specific manner to cleave flap strands. In addition, this enzyme has 5′ exonuclease activity. The enzyme has been characterized for its crystal structure, and its mutant has been used to investigate the substrate recognizing mechanisms. In addition, a Pyrococcus derived thermostable enzyme is known as a thermostable flap endonuclease.
Meanwhile, substrate specificity of these flap endonucleases has been utilized in recent years for analyzing genetic polymorphism.
By genetic polymorphism is meant a phenomenon that nucleotide sequences in the same position of a certain gene differ between different individuals, and it is distinguished from mutation by the frequency with which they occur. However, since genetic polymorphism may cause diseases directly, and single nucleotide polymorphism (SNP), the currently most frequent polymorphism, is thought to complicate lifestyle-related diseases and is considered a genetic predisposition, enormous data has been accumulated regarding the location and nucleotides of SNPs.
Known methods of SNP analysis using the flap endonuclease as stated above include an invader method. The method involves determining the presence/absence of a SNP by examining whether a flap endonuclease recognizes the three-nucleotide overlapping structure formed as a result of annealing of the target nucleic acid (SNP area on the genome) and invader and signal probes and cleaves the flap portion. However, flap endonucleases known to date have substrate specificity that is so broad that they detect genetic defects other than SNPs, such as nicks, and are insufficient in reliability.
[Non-patent Document 1] Kaiser, M., Lyamicheva, N., Ma, W., Miller, C., Neri, B., Fors, L., and Lyamichev, V., (1999) J. Biol. Chem., 274, 21387-21394
[Non-patent Document 2] Lyamichev, V., Brow, M. A. D., Varvel, V. E., and Dahlberg, J. E., (1999) Proc. Natl. Acad. Sci., 96, 6143-6148